The four serotypes of dengue virus cause an estimated 100-200 million cases human disease in tropical regions each year. Infection with one serotype produces durable immunity only to that type. Serotype is conferred by the viral structural proteins, capsid (C), pre-membrane (pre-M) and envelope (E). The dengue viral genome, an 11 kilobase strand of positive-sense RNA, contains the three structural protein genes followed by a series of 7 non-structural protein genes. Earlier, a full- length cDNA copy of the entire dengue type 4 virus (D4) genome was constructed. Transfection of mammalian cells with RNA transcripts from this template yielded D4 virus, designated D4 2A. We subsequently replaced the C-preM-E or preM-E genes of the full-length clone with the structural protein genes of two different strains of dengue type 2 virus (D2). In one construct the D4 preM-E genes were replaced with the preM-E genes of a D2 New Guinea C strain virus isolated from a human patient (D2 NGC parental) and in the other the C-preM-E or preM-E genes of the mouse- neurovirulent mutant selected from the parental virus during serial passage in mouse brain, i.e., D2 NGC neurovirulent. Neither D4 2A nor the chimeric virus (D2 ME parental)/D4 was neurovirulent in suckling mice, whereas chimeric viruses containing the C-preM-E or preM-E genes of D2 NGC neurovirulent produced fatal encephalitis. It appears that some or all of the genetic loci responsible for neurovirulence are located in the structural protein genes. Sequence analysis of the cloned C-preM-E genes of parental and neurovirulent D2 NGC identified 7 mutations in the neurovirulent mutant which result in an amino acid change. Based on this information a series of chimeric viruses were constructed in which single or multiple mutations in pre-M and/or E were substituted into the parental sequence, and the resulting mutants were tested for neurovirulence in mice. A mutation in the N-terminal one- third of E protein which changed the negatively charged glutamic acid in the parental E protein to a positively charged lysine appeared to be sufficient to produce the neurovirulence phenotype.